Structure and regulatory mechanism of Aquifex aeolicus NtrC4: variability and evolution in bacterial transcriptional regulation.
Genetic changes lead gradually to altered protein function, making deduction of the molecular basis for activity from a sequence difficult. Comparative studies provide insights into the functional consequences of specific changes. Here we present structural and biochemical studies of NtrC4, a sigma-54 activator from Aquifex aeolicus, and compare it with NtrC1 (a paralog) and NtrC (a homolog from Salmonella enterica) to provide insight into how a substantial change in regulatory mechanism may have occurred. Activity assays show that assembly of NtrC4's active oligomer is repressed by the N-terminal receiver domain, and that BeF3- addition (mimicking phosphorylation) removes this repression. Observation of assembly without activation for NtrC4 indicates that it is much less strongly repressed than NtrC1. The crystal structure of the unactivated receiver-ATPase domain combination shows a partially disrupted interface. NMR structures of the regulatory domain show that its activation mechanism is very similar to that of NtrC1. The crystal structure of the NtrC4 DNA-binding domain shows that it is dimeric and more similar in structure to NtrC than NtrC1. Electron microscope images of the ATPase-DNA-binding domain combination show formation of oligomeric rings. Sequence alignments provide insights into the distribution of activation mechanisms in this family of proteins.
Batchelor
Joseph D
JD
Graduate Group in Biophysics, Physical Biosciences Division, Lawrence Berkeley National Laboratory and the Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Doucleff
Michaeleen
M
Lee
Chul-Jin
CJ
Matsubara
Koshi
K
De Carlo
Sacha
S
Heideker
Johanna
J
Lamers
Meindert H
MH
Pelton
Jeffrey G
JG
Wemmer
David E
DE
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000648:ultrastructure; D019295:Computational Biology; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000378:metabolism; D019281:Dimerization; D019143:Evolution, Molecular; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D015534:Trans-Activators / Q000737:chemistry; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012996:Solutions; D015534:Trans-Activators; D000255:Adenosine Triphosphate